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免疫学信息网 http://www.immuneweb.com
Techniques to manipulate DNA
Phenol/chloroform extractionEthanol precipitationConcentration determinationGel ElectrophoresisDNA hybridizationSouthern BlottingMelting temperaturesPCR
免疫学信息网 http://www.immuneweb.com
BRCA3EcoRI EcoRI
NIH
Example: Is the BRCA3 gene involved in Suppressing breast cancer?
BRCA1
BRCA2
BRCA3
免疫学信息网 http://www.immuneweb.com
Paper Towels
Southern Blotting
1
tissue
SDSProtKPhenolChloro
ETOH ppt
Spin
免疫学信息网 http://www.immuneweb.com
The “Hyperchromic Shift”
2
免疫学信息网 http://www.immuneweb.com
Paper Towels
Southern Blotting
1
tissue
SDSProtKPhenolChloro
ETOH ppt
Spin
免疫学信息网 http://www.immuneweb.com
Agarose Gel Electrophoresis
_ +
3
免疫学信息网 http://www.immuneweb.com
Sample Well 25 ng 1 kb ladder0.8% Agarose
1
2
4
681012kb
Agarose Gel Electrophoresis
Sybergold Detection Limit: ~0.2-0.5 ng DNA
EtBr Detection Limit: ~5-10 ng DNA
(Not in handout)
免疫学信息网 http://www.immuneweb.com
Log (MW) is linearly related to mobility
4100 bp
Low % has noResolving powerat low MW
1 kb
10 kb
High % has noresolving powerat high MW
免疫学信息网 http://www.immuneweb.com
Separation Range Vs. % Agarose
5Low Geling Agarose 4-6 0.02-0.4
免疫学信息网 http://www.immuneweb.com
TEMED(N,N,N,N-tetramethylenediamine)
7
免疫学信息网 http://www.immuneweb.com
PolyAcrylamide Gels
8
免疫学信息网 http://www.immuneweb.com
DNA topology affects mobility of DNA
9
免疫学信息网 http://www.immuneweb.com
PFGE can resolve large DNA fragments
10
免疫学信息网 http://www.immuneweb.com
Paper Towels
Southern Blotting
1
tissue
SDSProtKPhenolChloro
ETOH ppt
Spin
免疫学信息网 http://www.immuneweb.com
Helix Formation: Nucleation and Propagation
11
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A-T
A-T
G-C
Helix Melting
12
免疫学信息网 http://www.immuneweb.com
What parameters affect theTm?
(Tm is defined as the temp at which half the molecules
are single-stranded)
免疫学信息网 http://www.immuneweb.com
Tm depends on DNA length
13
% d
sDN
ASigmoidalCurve:HighlyCooperativeMelting!
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Effect of % GC Content on Tm
14
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Effect of [Salt] on Tm
15
免疫学信息网 http://www.immuneweb.com
i) For oligos 4 - 18 nucleotides
Tm = (#A-T bp) 2 C + (#G-C bp) 4C
ii) For 14 – 70 nucleotides
0.01 M < [Na+] < 0.4 M, and 30 < %GC < 75
Tm = 81.5 + 16.6 log10[Na+] + 0.41 (% G + C)– 600/N – P
where N = # nucleotides, P = %mismatches
Predicting melting temperatures
(see lecture notes)
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Kinetics of Hybridization:Do I have time for a soda or a week-end in New York City?
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Cot Curves: Hybridization vs. Complexity
16
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A Bi-Phasic Cot Curve
17
5 kb Repeats (20% total)
100 kb fragment
Repeat DNA = 5 kbLongest non-repeat DNA = 100kb - 20 kb = 80 kbDifference in complexity = 80/5 = 16
免疫学信息网 http://www.immuneweb.com
Paper Towels
Southern Blotting
1
tissue
SDSProtKPhenolChloro
ETOH ppt
Spin
免疫学信息网 http://www.immuneweb.com
Results of Southern Blot
Control Tissue Cancer Tissue
BRCA3EcoRI EcoRI
NIH
免疫学信息网 http://www.immuneweb.com
BRCA3EcoRI EcoRI
NIH
免疫学信息网 http://www.immuneweb.com
94 C(30 s)
40-60 C(30 s)
72 C(30-60 s/kb)
Melt Anneal Extend
The Polymerase Chain Reaction (PCR)
1820-30X
免疫学信息网 http://www.immuneweb.com
94 C(30 s)
40-60 C(30 s)
72 C(30-60 s/kb)
Melt Anneal Extend
Cycle #2
NIH20-30X
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Results of PCR #1
Control Tissue Cancer Tissue
BRCA3EcoRI EcoRI
NIHPrimer Set #1
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Results of PCR#5
Control Tissue Cancer Tissue
BRCA3EcoRI EcoRI
NIHPrimer Set #5
免疫学信息网 http://www.immuneweb.com
PCR mutagenesis
EcoRIHindIII
Isolate products, mix
Amplify
digest, subclone sequence 20
PvuII
免疫学信息网 http://www.immuneweb.com
免疫学信息网 http://www.immuneweb.com
Linear Amplification
GeometricAmplification
19
免疫学信息网 http://www.immuneweb.com
hsp70
EcoRI EcoRI
NIH
Example: Is heat shock factor bound to promoters before heat shock?
EcoRI
HSE
HSF
免疫学信息网 http://www.immuneweb.com
Optimal separation of fragments > 2 kbrequires low voltage
0.5 10 V/cm
6